Novel gene having reverse transcriptas motif

ABSTRACT

The present invention aims to provide a novel gene having a reverse transcriptase motif. The invention isolates a novel gene having a reverse transcriptase motif, and gives its complete base sequence determined. The invention also provides a protein encoded by the gene, and an antibody against the protein. The use of them is useful in developing a method for detecting telomerase activity, a method for detecting a cancer cell, a telomerase activity inhibitor, and a method for screening a telomerase activity inhibitor.

TECHNICAL FIELD

This invention relates to a novel gene having a reverse transcriptasemotif, a protein encoded by the gene, an antibody against the protein,and a method for detecting reverse transcriptase activity, a method fordetecting a cancer cell, and a reverse transcriptase activity inhibitoror a method for screening a reverse transcriptase activity inhibitor,each of the methods and the inhibitor using any of the novel gene, theprotein, and the antibody.

The invention also relates to a method for diagnosing cancer by the useof the above detecting method, a cancer diagnostic agent containing aprobe complementary to the gene, and a cancer diagnostic agentcontaining the antibody.

BACKGROUND ART

Telomerase is an enzyme known to have the function of repairing thelength of telomere which is shortened by each cell division (Greider C.W. and Blackburn E. H., (1987) Cell, 51, 887-898; Morin G. B. (1989)Cell, 59, 521-529).

Most cancer cells show telomerase activity (Kim N. W. et al., (1994)Science, 206, 2011-2015), a finding strongly suggesting that telomerasetakes part in maintaining the infinite proliferation of cancer cells.

The measurement of telomerase activity, therefore, is important fordiagnosis of cancer, and a substance inhibiting telomerase activity canbe expected to serve as an anticancer drug causing few adverse reactionsto normal cells (Counter C. M. et al., (1989) EMBO J., 11, 1921-1929;Counter C. M. et al., (1994) Proc. Natl. Acad. Sci. USA, 91, 2900-2904;Chadenneau C. et al., (1995) Cancer Res., 55, 2533-2536; Hiyama E. etal., (1995) Nature Med., 1, 249-255; Shay J. W. et al., (1995) Mol.Cell. Biol., 15, 425-432).

One of conventional methods for measuring telomerase activity is tomeasure telomerase enzyme activity.

According to this method, a cell extract needs to be preparedbeforehand, with enzyme activity being maintained. Then, a telomereelongation reaction (telomerase reaction) is performed. The elongatedtelomere is measured for the amount of the resulting DNA, directly orafter amplification by polymerase chain reaction (PCR). This method wasnot a convenient, effective method for measuring telomerase activity.

Another method for measuring telomerase activity is to measure theexpression of a telomerase gene. Such a method requires theidentification of the gene correlated to telomerase activity.

As one of such genes, a gene encoding a protein having a reversetranscriptase motif with a molecular weight of about 130 kDa wasrecently isolated from mRNA derived from a human testis or a cancer cell(Meyerson M., et al., (1997) Cell, 90, 785-795; Nakamura T. M. et al.,(1997) Science, 277, 955-959).

Since the expression of this gene showed high correlation withtelomerase activity, this gene has been speculated to code for a humantelomerase catalytic subunit.

However, much is yet unknown about telomerase.

For example, it is still unknown whether a plurality of genes having areverse transcriptase motif exists, and whether a gene, or a pluralityof genes, showing telomerase activity exist. It is also unknown whetherthe telomerase activity of a normal germ cell and that of a cancer cellalways come from the same genetic product, and whether the telomeraseactivities of all cancer cells can be explained by a single gene.Solutions to these questions are desperately wanted in the fields ofcancer diagnosis and treatment focusing on telomerase.

DISCLOSURE OF THE INVENTION

The present invention aims to provide a novel gene encoding a proteinhaving a reverse transcriptase motif, the novel gene having correlationwith reverse transcriptase activity.

The invention also aims to provide a protein encoded by the gene, and anantibody against the protein.

The invention further aims to provide a method for measuring reversetranscriptase activity, and a method for detecting a cancer cell, eachmethod using the gene, the protein, or the antibody.

The reverse transcriptase as used herein refers collectively to nucleicacid polymerases which perform DNA synthesis using RNA as template. Aknown representative reverse transcriptase is that found inretroviruses.

Telomerase synthesizes a telomere DNA sequence by using its own subunit,single-stranded RNA, as template. Thus, telomerase is positioned as anassociate of RNA-dependent DNA polymerases, i.e., an associate ofreverse transcriptases.

In the present specification, reverse transcriptase activity refers,preferably, to telomerase enzyme activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is electrophoretic pattern photographs showing the correlationbetween telomerase activity and the expression of CRT-1 gene. In FIG. 1,PC93, COLO203, HCT-15, DU145, LNCap, PC3, MCF7, SC6, HL60, and WI-38denote, respectively, cell strains from prostatic cancer, colon cancer,colon cancer, prostatic cancer, prostatic cancer, prostatic cancer,mammary carcinoma, gastric cancer, promyelocytic leukemia, and a cellstrain derived from the lung. Testis denotes a testis (tissue).

BEST MODE FOR CARRYING OUT THE INVENTION

In the light of the aforementioned questions, the inventors of thepresent invention conducted extensive studies in an attempt to discovera novel gene having correlation with reverse transcriptase activity. Asa result, the inventor found out a novel gene encoding a protein havinga reverse transcriptase motif from a human promyelocytic leukemia cellstrain HL60 cell, and confirmed correlation between the expression ofthe resulting gene and reverse transcriptase activity. These findingshave led the inventor to accomplish this invention.

Therefore, the use of the gene according to the invention, a proteinencoded by the gene, or an antibody against the protein makes itpossible to measure reverse transcriptase activity, and to control theexpression of reverse transcriptase activity.

Furthermore, it becomes possible to design an inhibitor of the activity,or to develop a method for screening an inhibitor of reversetranscriptase activity.

More particularly, the invention provides a gene, a protein, anantibody, a method for detecting a cancer cell, a reverse transcriptaseactivity inhibitor, a method for screening the inhibitor, a reversetranscriptase activity regulator, a ribozyme, a reverse transcriptaseactivity suppressor, a method for screening a reverse transcriptaseactivity inducer, and a reverse transcriptase activity inducer, asdescribed below.

1. A CRT-1 gene comprising a base sequence set forth in SEQ ID NO: 1, 9or 11 in the Sequence Listing.

2. A gene encoding a protein comprising an amino acid sequence set forthin SEQ ID NO: 2, 10 or 12 in the Sequence Listing.

3. A gene encoding a protein comprising an amino acid sequence obtainedby substituting or deleting one or more amino acids of an amino acidsequence set forth in SEQ ID NO: 2, 10 or 12 in the Sequence Listing, orby adding one or more amino acids to an amino acid sequence set forth inSEQ ID NO: 2, 10 or 12 in the Sequence Listing,

-   -   wherein the protein has reverse transcriptase activity.

4. A gene which is hybridized under stringent conditions with the geneaccording to any one of 1 to 3 above, and which encodes a protein havingreverse transcriptase activity.

5. A protein comprising an amino acid sequence set forth in SEQ ID NO:2, 10 or 12 in the Sequence Listing.

6. A protein comprising an amino acid sequence obtained by substitutingor deleting one or more amino acids of an amino acid sequence set forthin SEQ ID NO: 2, 10 or 12 in the Sequence Listing, or by adding one ormore amino acids to an amino acid sequence set forth in SEQ ID NO: 2, 10or 12 in the Sequence Listing, the protein having reverse transcriptaseactivity.

7. An antibody against the protein according to any one of 5 and 6above.

8. An oligonucleotide comprising a base sequence of an antisense strandof the gene according to any one of 1 to 4 above.

9. A method for detecting a cancer cell, comprising detecting the geneaccording to any of 1 to 4 above.

10. A method for detecting a cancer cell, comprising using the antibodyagainst the protein according to 7 above.

11. A reverse transcriptase activity inhibitor having an oligonucleotidecomprising the base sequence according to any one of 1 to 4 above.

12. A method for screening a reverse transcriptase activity inhibitor,comprising using an oligonucleotide comprising the base sequenceaccording to any one of 1 to 4 above.

13. A method for detecting a cancer cell, comprising detecting the geneaccording to any one of 1 to 4 above.

14. A reverse transcriptase activity inhibitor having an oligonucleotidecomprising the base sequence according to any one of 1 to 4 above.

15. A method for screening a reverse transcriptase activity inhibitor,comprising using an oligonucleotide comprising the base sequenceaccording to any one of 1 to 4 above.

16. An agent which acts on the CRT-1 gene to regulate reversetranscriptase activity.

17. A ribozyme of the CRT-1 gene.

18. An intracellular antibody against the CRT-1 protein.

19. A reverse transcriptase activity suppressor comprising a dominantnegative protein of the CRT-1 protein.

20. A method for screening a reverse transcriptase activity inducer,comprising using the CRT-1 protein or gene.

21. A reverse transcriptase activity derivative comprising the CRT-1gene, the CRT-1 protein, or a mutant thereof.

22. A method for diagnosing cancer, comprising using the above methodfor detecting a cancer cell.

23. A cancer diagnostic agent comprising a probe complementary to thegene above.

24. A cancer diagnostic agent comprising the antibody above.

The present invention will now be described in more detail by referenceto its embodiments.

If a gene refers definitely to DNA obtained by reverse transcriptionfrom mRNA present in nature (DNA obtained by amplification of the DNA isincluded), the gene is called cDNA.

(Gene having a Reverse Transcriptase Motif)

A gene having a reverse transcriptase motif according to the presentinvention can be obtained by the following procedure:

(1) Prepare mRNA from a human-derived cell.

Concretely, HL-60 can be used preferably.

(2) Prepare total RNA from the selected cell.

In constituting such total RNA, a publicly known method can be usedpreferably.

(3) Then, prepare poly(A)+RNA(mRNA).

There are no restrictions on a method for preparing it, and a publiclyknown method or a commercially available kit can be used.

(4) Based on the resulting mRNA, construct a cDNA library of HL-60 foruse in 5′RACE and 3′RACE.

There are no restrictions on a method for preparing it, and a publiclyknown method or a commercially available kit can be used.

(5) No restriction is imposed in selecting primers for use in the 5′RACEmethod. However, primers for amplifying the 5′ region of a gene having areverse transcriptase-like motif can be designed based, for example, ona human-derived EST clone (GenBank Accession Number AA281296) havinghigh homology to a telomerase catalytic subunit of Ciliophora Euplotesor the yeast S. cerevisiae.

Concretely, primers used in Examples to be offered later on can bequoted.

Synthesis of primers can be achieved with the desired purity by anordinary method.

(6) There are no restrictions on the conditions for amplification ofgene, but optimal conditions for amplification can be selected bypublicly known methods.

A commercially available kit can also be used.

Concretely, the conditions for amplification are those used in theExamples.

(7) After purifying the resulting reaction product, subclone it into asuitable vector, and determine the base sequences of gene fragmentscloned.

There are no restrictions on the usable subcloning vector, butconcretely, the vector used in the Examples is preferred.

A commercially available kit can also be used.

There are no restrictions on a method for base sequencing, and apublicly known method, and a sequencer using it, can be used (e.g., Taqcycle sequencing, the method described in “Biotechniques, vol. 7”,(1989), pp. 494-499).

(8) The primers for the 3′RACE method can also be designed in the samemanner as the primer designing for 5′RACE.

Concretely, the sequences used in the Examples are cited.

As for the conditions for amplification, optimal conditions can beselected as with the 5′RACE method.

Concretely, the conditions used in the Examples can be cited.

A commercially available kit can also be used.

(9) Similarly purify the resulting reaction product, subclone it into asuitable vector, and determine the base sequences of the resultingclones.

(10) A gene of the total length can be yielded by designing primers,which are hybridized with the 5′-terminal and 3′-terminal of cDNA, fromthe gene fragments obtained by the 5′RACE and 3′RACE methods describedabove, and performing a suitable amplification reaction.

As such an amplification reaction, RT-PCR used in the Example can beused preferably.

There are no restrictions on a method for determining the base sequenceof the resulting gene, and a publicly known method, and a sequencerusing it, can be used.

(Detection of Tissue Distribution, Method for Detecting Cancer Cell,Method for Screening Anticancer Drug).

It is possible to prepare a probe with the use of a fragment of theresulting gene, and find out a tissue in which the gene is expressed.

For example, the probe can be labeled with a publicly known means oflabeling, and a publicly known northern blotting method can beperformed.

Also, the gene expressed in a particular tissue can be assayed by use ofa particular standard material.

Thus, telomerase activity can be confirmed by detecting the expressionof the gene.

If telomerase activity is related to the presence of a cancer cell,detection and assay of such gene provide a method for detecting a cancercell.

Similarly, quantitative determination of the expression of the geneprovides a method for ascertaining the effect of an anticancer drug.

In this case, the effect of the anticancer drug can be confirmed byinvestigating changes in the amount of gene expression depending onwhether the anticancer drug was administered or not, or changes in theamount of the gene expressed within a particular period of time afteradministration of the anticancer drug.

(Protein, Method for Detecting Cancer Cell)

The above-described method makes it possible to obtain a human-derivednovel gene having a reverse transcriptase motif according to theinvention, and identify the amino acid sequence of a protein encoded bythe gene from information on the base sequence of the gene.

That is, the protein encoded by the novel gene of the invention is aprotein comprising the amino acid sequence set forth in SEQ ID NO: 3 inthe Sequence Listing.

Furthermore, the protein of the invention is not restricted to thisamino acid sequence, and includes a protein obtained by substituting ordeleting one or more amino acids of the amino acid sequence, or byadding one or more amino acids to the amino acid sequence, the proteinhaving telomerase activity (i.e., a mutant protein).

Hence, examples of a polynucleotide encoding the protein havingtelomerase activity of the invention include not only a polynucleotidecomprising the base sequence set forth in SEQ ID NO: 1 in the SequenceListing, but also the same polynucleotide whose structure has beenpartly changed by spontaneous or artificial mutation without changingtelomerase activity, the main function of a polypeptide encoded by thepolynucleotide (i.e., a mutant gene).

Regarding methods for introducing such an artificial mutation, referenceshould be made, for example, to “Molecular Cloning 2nd Edition” (ColdSpring Harbor Laboratory Press, 1989) pp. 15.1-15.113.

Also, a gene to be obtained by replacing at least some bases of the basesequence of the gene by other types of bases as a result of degeneracyof the genetic codes can give a protein having the same amino acidsequence.

Therefore, the gene encoding the protein of the invention includes alldegeneracy patterns capable of encoding the protein of the invention andmutant proteins derived from the protein.

There are no restrictions on a method for obtaining the protein of theinvention (including mutant proteins).

Examples of the method are various methods, such as a method ofartificial peptide synthesis by a protein synthesizer or the like, andmethods for expressing proteins by genetic engineering on the basis ofinformation on the base sequence of the gene encoding the proteinobtained by the invention.

The CRT-1 protein may be expressed alone or as a fusion protein fused toMBP (maltose binding protein) or the like, or may be expressed in a formhaving a tag, such as FLAG peptide, added thereto.

The genetic engineering technique enables the protein to be prepared byuse of bacteria, such as E. coli, or yeast, or animal cells or insectcells.

An example is a method for obtaining a transformant by selecting asuitable vector and a suitable host, and introducing the gene (forexample, this can be achieved by the method described in “CellEngineering Protocol”, Shujun-Sha, 1991, pp. 105-107).

The resulting transformant is cultured to amplify the gene and express aprotein, whereby the desired-protein can be expressed.

Then, the cultures are collected, and if desired, concentrated,solubilized, dialyzed, and variously chromatographed, whereby theprotein of the invention and mutant proteins therefrom can be obtained.

For the culturing of the transformant, various textbooks are available.For instance, the method described in “Methods of Microbial Experiments”(the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1992) can beemployed.

The expression of the desired protein (including mutant proteins) basedon the base sequence described in the invention can be carried out by apublicly known method.

For this purpose, the host may be any of bacteria such as E. coli,yeast, and animal cells. Particularly, animal cells are preferred.

To incorporate the gene into a cell, the liposome method, theelectroporation method, etc. can be used.

The use of the DEAE-dextran method (Pharmacia), in particular, ispreferred.

Purification methods for purifying a protein from the resulting cultureinclude immunoprecipitation, salting-out, ultrafiltration, isoelectricprecipitation, gel filtration, electrophoresis, ion exchangechromatography, various affinity chromatographic techniques such ashydrophobic chromatography and antibody chromatography,chromatofocusing, adsorption chromatography, and reverse phasechromatography. Any of these methods may be selected suitably.

In the manufacturing stage, the desired protein may be produced by atransformant as a fusion peptide fused to other polypeptide.

If desired, the fusion product may be treated with a chemical substance,such as cyanogen bromide, or an enzyme, such as protease, in thepurification step to cut out the desired protein.

(Antibody, Method for Detecting Cancer Cell by Use of Antibody)

A method for obtaining an antibody against all of or a part of theresulting protein (including mutant proteins) may be a publicly knownmethod.

The antibody of the invention includes a polyclonal antibody and amonoclonal antibody, as long as each of the antibodies reacts with theprotein of the invention (or its mutant).

Its active fragment, and a chimeric antibody containing the activefragment are also included.

An antibody, i.e., an immunoglobulin, has an H chain and an L chain, andis classified into five classes (IgA, IgD, IgE, IgG, IgM) byphysicochemical properties and immunological properties.

Of these classes, IgA and IgG are further divided into subclasses by thetype of the H chain.

The novel antibody of the invention includes those belonging to allthese classes and subclasses.

Furthermore, the antibody of the invention does not necessarily need tobe the entire antibody molecule, but may be a part of the molecule(active fragment), if it has activity.

Examples of the active fragment are F(ab′)₂, Fab′, Fab, Fv, recombinantFv, and single-stranded Fv.

For example, decomposition with pepsin gives F(ab′ 2 and Fc′, whiledecomposition with papain gives Fab and Fc.

These active fragments may be used alone, but if desired, may be used asa new complex formed by their joining to substances such as albumin orpolyethylene glycol.

Generally, such a complex shows its effect to maximum in vivo withoutbeing decomposed for a long time.

Methods for performing the addition of a substance, such as albumin orpolyethylene glycol, to an active fragment are described, for example,in “Antibodies, A Laboratory Manual” (Cold Spring Herber Laboratory,1988), pp. 77-81 and pp. 129-137.

Generally, the active fragment can be easily bound to albumin or thelike by using a bifunctional reagent, such as SPDP (Pharmacia), SMPB(Pias) or EMCS (Dotite).

For a method for preparing the antibody of the invention, reference canbe made, for example, to “Method for Handling Immunological Experiments”(edited and published by The Japanese Society for Immunology).

An immunogen may be a part of the protein of the invention, i.e., theamino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing,or a polypeptide comprising 8 or more consecutive amino acids of theamino acid sequence of the mutant protein.

If the protein as the immunogen has been purified to such a degree thatit can be used for the preparation of an antibody, the protein may beobtained by whatever method.

If the immunogen is a polypeptide comprising 8 to about 20 amino acids,it may be bound to a carrier such as keyhole-limpet hemocyanin (KLH) foruse as an antigen.

An animal to be immunized with the immunogen may be any animal otherthan human, and it is preferred to select an animal species, capable ofproducing the desired antibody, from animals usually used among peopleskilled in the art.

The polyclonal antibody is obtained by purifying the resultingantiserum.

Purification may be performed by combining methods, such as salting-out,ion exchange chromatography, and affinity chromatography.

The monoclonal antibody is obtained by obtaining a fused cell by anordinary method for preparing a hybridoma, and then causing the fusedcell to produce an antibody.

For cell fusion, a technique using polyethylene glycol, Sendai virus, orelectric pulses is usable.

In addition to the above methods, a genetic engineering method may beused to obtain the monoclonal antibody.

For example, mRNA is collected from splenic cells or lymphocytes of ananimal immunized with the protein of the invention or a part of theprotein, or from a hybridoma which produces the monoclonal antibody, anda cDNA library is constructed based on the mRNA.

Then, antibodies are expressed from the cDNA library.

Clones producing the antibodies reactive with the antigen are obtainedfrom the cDNA library by screening. The resulting clones are cultured,and the desired antibody can be purified from the culture mixture by acombination of methods, such as salting-out, ion exchangechromatography, and affinity chromatography.

The resulting antibody of the invention can be used to detect telomerasewhich is present in various tissues.

In detecting it, western blotting, a usually employed method, can beused preferably.

The antibody can be used in preparing an antibody column, which is usedfor purifying the protein of the invention, its mutant, or a part of theprotein or the mutant. The antibody is also usable in detecting theprotein, its mutant, or a part of the protein or the mutant which iscontained in respective fractions.

Furthermore, the use of a particular standard material makes it possibleto assay telomerase expressed in a particular tissue.

Thus, telomerase activity can be confirmed by detecting the expressionof the telomerase.

If telomerase activity is related to the existence of a cancer cell,detection and assay of such telomerase provide a method for detecting acancer cell.

Similarly, quantitative analysis of the expression of the telomeraseprovides a method for ascertaining the effect of an anticancer drug.

In this case, the effect of the anticancer drug can be confirmed byinvestigating changes in the amount of telomerase expression dependingon whether the anticancer drug was administered or not, or changes inthe amount of telomerase expression within a particular period of timeafter administration of the anticancer drug.

As another clinical application, diagnosis of cancer can be made, forexample, by taking an extract of a cell or a pathological tissue as aspecimen, and examining the presence or absence of the gene of theinvention therein.

Concretely, there is a method which uses a sequence complementary to thegene as a probe to examine the presence or absence of the gene.

The length of the sequence used as the probe is 10 to 1,300 bases,preferably 10 to 1,000 bases, more preferably 20 to 400 bases.

In examining the presence or absence of the gene, the gene may be orneed not be amplified. As a means of amplification, RT-PCR, or TMA(transcription mediated amplification, Japanese Laid-Open PatentApplication, Hei 4-500759) can be used.

As a means of detection, HPA (hybridization protection assay, JapaneseLaid-Open Patent Application, Hei 2-5043147) can be used.

As a concrete method for diagnosis of cancer, the method described, forexample, in Japanese Laid-Open Patent Application, Hei9-502102 or U.S.Pat. No. 5,489,508 can beused.

Particularly if the material used is a pathological tissue, in situhybridization using the aforementioned probe, for example, is usable.

It is also possible to use ABC tissue staining which uses the antibodyof the invention.

(Antisense Oligonucleotide, Telomerase Activity Inhibitor, Method forScreening Telomerase Activity Inhibitor)

An antisense polynucleotide based on the above-mentioned gene obtainedby the invention includes all of a plurality of nucleotides boundtogether, which comprise bases, phosphates and sugars, and those whichare naturally nonexistent.

Typical of them are DNA and mRNA.

The antisense polynucleotide derivatives of the invention include thosewhich are similar to polynucleotides in stereostructure and function.

Examples are those in which other substances have been bound to the3′-terminal or 5′-terminal of polynucleotides, polynucleotides in whicha modification, such as substitution, deletion or addition, has occurredin at least some of the bases, sugars or phosphates, those having bases,sugars or phosphates which are not naturally existent, and those havinga skeleton other than a sugar-phosphate skeleton.

The antisense polynucleotide and its derivatives may be those which canbe hybridized with the gene of the invention and any portions of itsmutant gene.

The antisense polynucleotide and its derivatives can be used as aresearch polynucleotide probe for investigating the existence orexpression status of the gene encoding the protein or its mutant of theinvention contained in a tissue or cell.

They are also usable as polynucleotide probes for diagnosis.

As probes, those which have 12 or more bases and a GC content of 30 to70% are preferred, and those which have 16 or more bases and a GCcontent of 30 to 70% are particularly preferred.

By using the antisense polynucleotide and its derivatives, theexpression of the protein (including its mutant) of the invention can beregulated.

This is because they can be expected to suppress the expression of theabove protein by their hybridization with the gene or mRNA coding forthe protein. Thus, they can be used as therapeutics for diseases, suchas cancer, based on the function the protein is involved in, i.e.,telomerase activity.

That is, antisense drugs can be developed from the antisensepolynucleotide and its derivatives.

Generally, a method which regulates the expression of a polypeptide byusing a polynucleotide containing a base sequence complementary to DNAor mRNA encoding the polypeptide is called the antisense method.

The polynucleotide having a complementary sequence is believed to bindto DNA or mRNA, a bearer of genetic information, somewhere among thefollowing stages, {circle over (1)} transcription from gene to pre-mRNA,{circle over (2)} processing from pre-mRNA to mature mRNA, {circle over(3)} nuclear membrane passage, and {circle over (4)} translation toprotein, to affect the normal flow of genetic information transduction,thereby regulating the expression of polypeptide.

Generally, a base sequence containing 15 or more bases is considered tobe a sequence having specificity (Yokoyama, K., Protein, Nucleic Acidand Enzyme, vol. 38, 754-765, 1994).

Thus, if the antisense polynucleotide and antisense polynucleotidederivative of the invention contain a base sequence complementary tomRNA for the protein and its mutant of the invention, and comprising 15or more bases, they are speculated to bind specifically to the gene ofthe invention or mRNA for the gene of the invention.

In incorporating the polynucleotide into a cell, too large a length ofthe polynucleotide is not suitable.

The antisense polynucleotide or its derivative of the invention may beof any length. However, if it is considered that the antisensepolynucleotide of the invention or its derivative is to be taken into acell to regulate the expression of protein, the antisense polynucleotideor the antisense polynucleotide derivative preferably has a basesequence complementary to mRNA for the gene, the base sequencecomprising 15 to 30 bases, preferably 15 to 25 bases, more preferably 18to 22 bases.

As the antisense polynucleotide or the antisense nucleotide derivativeof the invention, various derivatives, i.e., various polynucleotidederivatives having a high power to bind to the desired DNA or mRNA, hightissue selectivity, high cell permeation, high nuclease resistance, andhigh intracellular stability, can be obtained by publicly knownantisense technologies with the aim of enhancing the effect of thepolynucleotides as drugs.

In regard to the ease of hybridization, it is generally consideredrecommendable to design a polynucleotide or a polynucleotide derivativewhich has a base sequence complementary to the base sequence of a regionwhere a stem loop is formed (“Clinical Immunology, vol. 25”, 1200-1206,1993).

The polynucleotide and its derivative of the invention can, if desired,form a stem loop.

A polynucleotide having a sequence complementary to the sequence nearthe initiation codon, ribosome binding site, capping site, or splicesite can generally be expected to be highly effective in suppressingexpression (“Japanese Journal of Cancer and Chemotherapy, vol. 20, No.13”, pp. 1899-1907).

Thus, the polynucleotide or polynucleotide derivative of the invention,which has a sequence complementary to the sequence near the initiationcodon, ribosome binding site, capping site, or splice site of the geneencoding the protein or its mutant of the invention, or mRNA for thegene, can be expected to have a high effect of suppressing expression.

Derivatives, which are now generally known, are preferably derivativeswith at least one of nuclease resistance, tissue selectivity, cellpermeation, and binding force being enhanced. Particularly preferredpolynucleotide derivatives are shown to be derivatives having aphosphorothioate bond (see “Japanese Journal of Cancer andChemotherapy”, vol. 20, No. 13, 1899-1907, 1993) as a skeletalstructure.

The polynucleotide and its derivatives of the invention also includederivatives having these functions or the above structure.

In connection with a method for producing the antisense polynucleotidederivative of the invention, it is possible to use, for example, themethod described in “Antisense Research and Applications” (Michael J.GAIT, pp. 290-299, CRC Publishing, Florida, 1993).

In the case of native DNA or RNA, for example, it can be synthesized bymeans of a chemical synthesizer, or the antisense polynucleotide of theinvention can be obtained by PCR using the gene encoding the protein ofthe invention as template.

Of the methylphosphonate type or phosphorothioate type derivatives, somecan be synthesized by a chemical synthesizer (e.g., Model 394 ofPerkin-Elmer Japan).

In this case, the procedure is performed in accordance with an operatingguide attached to the chemical synthesizer, and the resulting syntheticproduct is purified by HPLC using reverse phase chromatography, wherebythe desired polynucleotide or polynucleotide derivative can be obtained.

APPLICATION EXAMPLES

As generally explained above, gene therapy and various otherapplications can be achieved by using the whole or part of the gene ofthe invention, the protein encoded thereby, and their mutants. Someexamples of these applications will be explained in detail below.

(1) Example of Application to Gene Therapy

By expressing the antisense RNA of the invention intracellularly,translation into CRT-1 protein can be inhibited.

For this purpose, the total length of pCRT-1 or part of cDNA isincorporated into a cloning site of an expression vector for a suitableanimal cell in the reverse direction, whereby antisense RNA can beprepared with the use of a suitable promotor.

The above material of the invention is also applicable as anintracellular antibody which suppresses the activity of pCRT-1 protein.

Concrete examples are HIV therapy (Marasco W. A., Gene Therapy (1997) 4,11-15) and breast cancer therapy (wright M., et al., Gene Therapy (1997)4, 317-322), as already reported.

The monoclonal antibody against CRT-1 can be isolated by the hybridomamethod or the antibody library method.

The monoclonal antibody that can suppress the activity of CRT-1 isselected, and then cDNA encoding the variable region of the antibody canbe isolated from hybridoma cells.

The intracellular antibody can be constructed based on the constructionof a single-stranded antibody.

That is, variable regions of the H chain and the L chain are joinedtogether by a linker sequence, e.g., a 15-amino acid sequence ((fourGly's and Ser)x3), and the joining product can be expressedintracellularly by a suitable expression vector.

When a mutant of CRT-1 is expressed in a cell as a dominant negativemutant, it can suppress the activity of CRT-1.

The dominant negative mutant is constructed, for example, based on amutant which binds to a telomere sequence, but which is deficient in DNAsynthesizing ability.

The dominant negative mutant shows competitive inhibition of normalCRT-1.

It is also possible to inhibit CRT-1 by introducing into a cell aribozyme capable of specifically decomposing the mRNA of CRT-1.

A ribozyme is an RNA molecule with the activity of excising RNAsequence-specifically, and is known as a gene therapy of cancer or HIV(Looney D. and Yu M., Methods in Molecular Biology (1997) 74, 469-486;Duarte E. A., et al., Methods in Molecular Biology (1997) 74, 459-468).

(2) Applicability to Identification of an Intracellular FactorInteracting with CRT-1

Studies of telomerase in Tetrahymena and yeast have suggested thattelomerase in the cell forms complexes with a plurality of proteinfactors (Collins K., et al., Cell (1995) 81, 677-686; Linger J., et al.,Science (1997) 276, 561-567).

The factors interacting with CRT-1 can be isolated, for example, by thetwo-hybrid method, a publicly known technique using yeast (Cowell I. G.,Method in Molecular Biology (1997) 69, 185-202).

(3) CRT-1 Protein can be Provided as a Research Material, inCrystallized Form, for Structural Analysis, and can also be used in DrugDesigning.

The protein can also be used as a material for reconstitution of anenzyme system having telomerase activity.

Such a reconstitution system is usable in searching for a substancewhich regulates telomerase activity.

The present invention will now be described with reference to Examplesto be offered below. However, the invention is not restricted by theseExamples.

Example 1

cDNA Cloning

(1) Preparation of HL-60 mRNA and Construction of cDNA Library

Total RNA was prepared from HL-60 strain in accordance with the methodof Chirgwin et al. (1979) (Biochemistry 18, 5294-5299), and poly A+ RNAwas prepared with the use of mRNA Purification Kit (Pharmacia) inaccordance with the manufacturer's instructions.

HL-60 cDNA libraries for 5′RACE and 3′RACE were constructed with the useof Marathon™ cDNA Amplification Kit (CLONTECH) in accordance with themanufacturer's instructions.

(2) 5′RACE Method

Primers for amplifying the 5′ region of a gene having a reversetranscriptase-like motif, i.e., hTRT5 (sequence:5′-ccgctcgtagttgagcacgctgaa-3′) and telo-rev (sequence:5′-accctcttcaagtgctgtc-3′) were designed based on a human-derived ESTclone (GenBank Accession Number AA281296) having high homology to atelomerase catalytic subunit of Ciliophora Euplotes or the yeast S.cerevisiae.

These primers were obtained from Sawady Technology (as were the primersto be used below).

Using Takara LA PCR™ Kit Ver. 2 (Takara Shuzo), gene amplification wasperformed under the following conditions:

A reaction mixture (50 μl) was prepared to have a composition comprising1×LA PCR Buffer II (Mg²⁺), 0.2 mM dNTP, 0.2 μM of the hTRT5 primer andAP1 primer (attached to Marathon™ cDNA Amplification Kit), 2 μl of thecDNA library for 5′RACE, and 5U Takara LA Taq. The reaction mixture wasreacted with the use of GeneAmp PCR System 2400 of Perkin-Elmer/ABIunder the following conditions: 94° C. 1 min; 30 cycles of 94° C. 15sec, 68° C. 3 min, and 68° C. 7 min.

The reaction product was diluted 1:50. With 1 μl of this dilution astemplate, and using 0.2 μM of the telo-rev primer and AP2 primer(attached to Marathon™ cDNA Amplification Kit), the reaction wasperformed under the following conditions: 94° C. 1 min; 15 cycles of 94°C. 15 sec, 68° C. 3 min, and 68° C. 7 min.

The composition of the reaction mixture, other than the primers and thetemplate DNA, was the same as that of the former reaction mixture.

The reaction product at the second stage was purified with PCRPurification Kit (QIAGEN), and subcloned into a pGEM-T vector (Promega).The base sequences of the resulting clones were determined usingAmpliTaq FS Prism ready reaction cycle sequencing Kit(Perkin-Elmer/ABI).

As a result, a gene having a reverse transcriptase-like motif wasidentified.

The 3′-terminal 213 bp portion of an isolated sequence had the samesequence as that of hEST2/hTRT1 (Meyerson M. et al., (1997) Cell 90,785-795; Nakamura T. M. et al., (1997) Science 277, 955-959), which hadbeen isolated earlier as a gene having a reverse transcriptase-likemotif, but the portion upstream from the 214 bp portion on the3′-terminal side had an entirely different structure.

(3) 3′RACE Method

Primers for 3′RACE, i.e., hRT1 (sequence:5′-tgcgtttcctgccgagtgtgtgttgatcc-3′), hRT3 (sequence:5′-tgcacagatgaagatgtggagactcacgag-3′) and telo-for (sequence:5′-agttcctgcactggctgatgagtg-3′), were designed based on the sequences ofthe five fragments obtained by the 5′RACE method.

3′RACE was performed under the following conditions as was 5′RACE:

Using Takara LA PCR™ Kit Ver. 2, gene amplification was performed underthe following conditions:

A reaction mixture (50 μl) was prepared to have a composition comprising1×LA PCR Buffer II (Mg²⁺), 0.2 mM dNTP, 0.2 μM of the hRT1 or hRT3primer and AP1 primer, 2 μl of the cDNA library for 3′RACE, and 5UTakara LA Taq. The reaction mixture was reacted under the followingconditions: 94° C. 1 min; 30 cycles of 94° C. 15 sec, 68° C. 3 min, and68° C. 7 min.

The reaction product was diluted 1:50. With 1 μl of this dilution astemplate, and using 0.2 μM of the telo-for primer and AP2 primer, thereaction was performed under the following conditions: 94° C. 1 min; 15cycles of 94° C. 15 sec, 68° C. 3 min, and 68° C. 7 min.

The composition of the reaction mixture, other than the primers and thetemplate DNA, was the same as that of the initial reaction mixture.

The reaction product at the second stage was purified with PCRPurification Kit, and subcloned into a pGEM-T vector. The base sequencesof the resulting clones were determined using AmpliTaq FS Prism readyreaction cycle sequencing Kit.

(4) Acquisition of Gene having Region Encoding Amino Acids by RT-PCR

From the gene fragments obtained by the 3′RACE method, primer hCRT-rev(sequence: 5′-aagatgaagtctcactctgttgcccaggctggagtg-3′) and primerhCRT-rev2 (sequence: 5′-ctgaaaaact catatattca gtattttact cccacag-3′)were designed as primers to be hybridized with the 3′-terminal of cDNA.Using these primers and hRT3, and with cDNA library for RACE astemplate, a gene comprising a region encoding amino acids was obtained.

The reaction conditions were as follows:

A reaction mixture (50 μl) was prepared to comprise 1×LA PCR Buffer II(Mg²⁺), 0.2 mM dNTP, 0.2 μM of the hCRT-rev primer (or hCRT-rev2 primer)and hRT3 primer, 2 μl of the cDNA library for RACE, and 5U Takara LATaq. The reaction mixture was reacted with the use of GeneAmp PCR System2400 of Perkin-Elmer/ABI under the following conditions: 94° C. 1 min;30 cycles of 94° C. 15 sec, 68° C. 3 min, and 68° C. 7 min.

The resulting reaction product was subcloned into a pGEM-T vector, andthe base sequences of the resulting clones were determined usingAmpliTaq FS Prism ready reaction cycle sequencing Kit(Perkin-Elmer/ABI).

Several types of splicing variants were obtained, and sequences havingpossible reading frames for reverse transcriptase-like proteins werespeculated from them.

When the primer hCRT-rev was used, two types of the above base sequenceswere obtained.

One of the base sequences, and an amino acid sequence deduced therefromare shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the Sequence Listing.

The other base sequence, and an amino acid sequence deduced therefromare shown in SEQ ID NO: 9 and SEQ ID NO: 10 in the Sequence Listing.

The base sequence obtained through the use of the primer hCRT-rev2, andan amino acid sequence deduced therefrom are shown in SEQ ID NO: 11 andSEQ ID NO: 12 in the Sequence Listing.

The amino acid sequence of SEQ ID NO: 10 was the amino acid sequence ofSEQ ID NO: 2 having Leu inserted at the 361st position.

The amino acid sequence of SEQ ID NO: 12 was the amino acid sequence ofSEQ ID NO: 2 in which Leu had been inserted at the 361st position, Argat the 437th position had been substituted by Ser, and amino acidresidues had been further added.

Example 2

Genetic Diagnosis by RT-PCR Method

1. Analysis of Expression Distribution

Using primers (hRT3 and hTRT5) which amplify the gene CRT-1 having areverse transcriptase-motif according to the invention, the expressionof hCRT in cancer cell strains and testis having telomerase activity,and a normal diploid cell strain WI-38 having no telomerase activity wasinvestigated by RT-PCR.

RT-PCR was performed by the following procedure:

Total RNA was prepared from various cultured cells in accordance withthe guanidine isothianate/phenol method (Chomc zynski and Sacchi (1987)Anal. Biochem. 162,156-159), and cDNA was synthesized with the use ofFirst-Strand cDNA Synthesis Kit (Pharmacia) in accordance with theattached instruction manual.

Using 1/50 of the reaction product as template, PCR was performed underthe following conditions:

The reaction was to be carried out on a 20 μl scale, and a reactionmixture was prepared to comprise 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5mM MgCl₂, 0.2 μM of primers hRT3 and hTRT5, and 1U AmpliTaqGold(Perkin-Elmer).

Using GeneAmpPCR System 9600 of Perkin-Elmer/ABI, the reaction wasperformed under the conditions: 94° C. 10 min; 40 cycles of 94° C. 30sec, 55° C. 30 sec, and 72° C. 1 min.

The reaction product was analyzed by 1% agarose electrophoresis.

The expression of hCRT gene was observed in the cancer cell strains andthe testis, showing the expression of this gene to be correlated withtelomerase activity.

As positive controls for the reverse transcription reaction, the resultsof RT-PCR of G3PDH (glycer aldehyde 3-phosphate dehydrogenase) gene areshown in FIG. 1.

INDUSTRIAL APPLICABILITY

The present invention isolates a novel gene having a reversetranscriptase motif, and provides its whole base sequence determined.The invention also provides a protein encoded by the gene, and anantibody against the protein.

Such base sequence information, protein and antibody can be usedeffectively in developing a method for detecting telomerase activity, amethod for detecting a cancer cell, a telomerase activity inhibitor, anda method for screening a telomerase activity inhibitor.

1-15. (canceled)
 16. An antibody against a protein selected from thegroup consisting of: a protein comprising the amino acid sequence of SEQID NO: 2; a protein comprising an amino acid sequence containing oneamino acid substitution, deletion or addition as compared to SEQ ID NO:2, wherein the protein has reverse transcriptase activity; a proteincomprising the amino acid sequence of SEQ ID NO: 10; a proteincomprising an amino acid sequence containing one amino acidsubstitution, deletion or addition as compared to SEQ ID NO: 10, whereinthe protein has reverse transcriptase activity; a protein comprising theamino acid sequence of SEQ ID NO: 12; and a protein comprising an aminoacid sequence containing one amino acid substitution, deletion oraddition as compared to SEQ ID NO: 12, wherein the protein has reversetranscriptase activity.
 17. A cancer diagnostic agent comprising theantibody of claim 16.